Compositions and Methods for Assessing DNA Damage in a Library and Normalizing Amplicon Size Bias

20240026431

18/473564

September 25, 2023

Described herein are standards and methods of normalizing amplicon size bias. These standards may comprise unique molecular identifiers. In some embodiments, the standards and methods are for use with next generation sequencing (NGS) assays. Also described herein are methods for quantifying DNA damage in a sample comprising DNA using fluorescence or for determining the presence of DNA damage in a library.

Illumina, Inc. (San Diego, CA, US)

1. A pool of nucleic acid standards of different lengths, wherein the nucleic acid standards comprise a unique molecular identifier (UMI) and: a. a 5′ universal oligonucleotide, wherein the 5′ universal oligonucleotide is the same for all standards; b. a 3′ universal oligonucleotide, wherein the 3′ universal oligonucleotide is the same for all standards; and c. at least one region between the UMI and the 5′ universal oligonucleotide and/or between the UMI and the 3′ universal oligonucleotide; wherein the length of the at least one region determines the length of the standard.

2. The pool of standards of claim 1, wherein the pool further comprises a further nucleic acid standard that comprises a UMI and: a. a 5′ universal oligonucleotide, wherein the 5′ universal oligonucleotide is the same for all standards; and b. a 3′ universal oligonucleotide, wherein the 3′ universal oligonucleotide is the same for all standards; wherein the further nucleic acid standard does not comprise at least one region between the UMI and the 5′ universal oligonucleotide or between the UMI and the 3′ universal oligonucleotide.

3. The pool of standards of claim 1, wherein the at least one region between the UMI and the 5′ universal oligonucleotide and/or between the UMI and the 3′ universal oligonucleotide comprise 0.2 kb-10 kb.

4. The pool of standards of claim 1, wherein the 5′ universal oligonucleotide and/or the 3′ universal oligonucleotide each comprise an amplicon amplified from a sequence of interest.

5. The pool of standards of claim 1, wherein the at least one region between the UMI and the 5′ universal oligonucleotide and/or between the UMI and the 3′ universal oligonucleotide each comprise an amplicon amplified from a sequence of interest.

6. The pool of standards of claim 1, wherein the least one region between the UMI and the 5′ universal oligonucleotide and/or between the UMI and the 3′ universal oligonucleotide each comprise an arbitrary sequence.

7. A pool of nucleic acid standards of different lengths, wherein the nucleic acid standards comprise a UMI and: a. a 5′ partially overlapping oligonucleotide, wherein the 5′ partially overlapping oligonucleotide is identical over at least a portion of its sequence for all the standards; and/or b. a 3′ partially overlapping oligonucleotide, wherein the 3′ partially overlapping oligonucleotide is identical over at least a portion of its sequence for all the standards; wherein the lengths of the 5′ partially overlapping oligonucleotide and/or the 3′ partially overlapping oligonucleotide determines the length of the standard.

8. The pool of standards of claim 1, wherein the standards are double-stranded.

9. The pool of standards of claim 1, wherein the standards comprise double-stranded DNA.

10. The pool of standards of claim 1, wherein each standard comprises a different UMI.

11. The pool of standards of claim 1, wherein the UMIs comprised in the pool of standards are a random set of sequences comprising 16-20 base pairs.

12. The pool of standards of claim 11, wherein the UMIs comprised in the pool of standards are a random set of sequences comprising 18 base pairs.

13. The pool of standards of claim 1, wherein the pool of standards comprises 1×1010 or greater, 10×1010 or greater, or 100×1010 or greater standards, wherein each standard comprises a different UMI.

14. The pool of standards of claim 1, wherein the number of standards in the pool is greater than the number of amplicons generated by an amplification reaction.

15. A pool of standards, wherein: a. at least a first portion of the standards are from claim 1; and b. at least a second portion of the standards are a pool of nucleic acid standards of different lengths, wherein the nucleic acid standards comprise a UMI and: i. a 5′ partially overlapping oligonucleotide, wherein the 5′ partially overlapping oligonucleotide is identical over at least a portion of its sequence for all the standards; and/or ii. a 3′ partially overlapping oligonucleotide, wherein the 3′ partially overlapping oligonucleotide is identical over at least a portion of its sequence for all the standards, wherein the lengths of the 5′ partially overlapping oligonucleotide and/or the 3′ partially overlapping oligonucleotide determines the length of the standard.

16. A method of generating a pool of nucleic acid standards comprising: a. providing multiple copies of at least one sequence of interest comprising nucleic acids; b. providing a collection of oligonucleotides each comprising a UMI; c. providing a collection of insertion oligonucleotides of varying lengths; and d. ligating at least one sequence of interest of (a), at least one oligonucleotide comprising a UMI of (b), and at least one insertion amplicon of (c) to produce multiple nucleic acid standards of the pool of nucleic acid standards.

17. A method of generating a pool of nucleic acid standards comprising: a. providing multiple copies of at least one sequence of interest comprising nucleic acids; b. providing a collection of oligonucleotides each comprising a UMI; and c. ligating at least one sequence of interest of (a) and at least one oligonucleotide comprising a UMI of (b).

18. A method of normalizing amplicon size bias comprising: a. combining a sample comprising a target nucleic acid with a pool of nucleic acid standards of different lengths, wherein each standard comprises a UMI; b. amplifying the standards and amplicons of a sequence of interest comprised in the target nucleic acid; c. sequencing the standards and the amplicons of the sequence of interest to generate sequencing data; d. determining a bias profile based on amplicon size using sequencing data from the standards; and e. normalizing amplicon size bias using the bias profile.

19. A method of determining the presence of DNA damage in a library comprising one or more library molecule, wherein each library molecule comprises a double-stranded DNA insert with a hairpin adapter at each end of the insert, comprising: a. denaturing the first stand and second strand of the double-stranded DNA inserts comprised in library molecules; b. annealing a forward primer and a reverse primer to library molecules; c. amplifying to produce library amplicons; and d. assessing the presence of DNA damage based on the number of library amplicons produced.

20. A method of quantifying DNA damage in a sample comprising DNA using fluorescence comprising: a. combining: i. an aliquot of a sample comprising DNA, ii. one or more DNA repair enzyme; and iii. dNTPs, wherein one or more dNTP is fluorescently labeled; b. preparing repaired DNA; c. dephosphorylating the phosphates from dNTPs; d. binding the repaired DNA to carboxylate or cellulose beads; e. eluting the bound repaired DNA from the carboxylate or cellulose beads with a resuspension buffer; and f. measuring fluorescence of the repaired DNA to determine the amount of DNA damage.

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